(1) Field of the Invention
This invention relates generally to the regulation of apoptosis and, more particularly, to a method for inhibiting apoptosis using the Adenovirus RID protein and to applications of this method, including promoting survival of tissue transplants, treating autoimmune disease, and promoting tumor destruction in cancer patients.
(2) Description of the Related Art
Apoptosis, or programmed cell death, plays a fundamental role in regulation of the immune system. For review, see White, E. Genes & Development 10:1-15, 1996; van Parijs. L. and Abbas, A. K., Curr. Opin. Immunol. 8:355-361, 1996; Nagata, S., Cell 88:355-365, 1997. In recent years researchers have shown that some members of the tumor necrosis factor (TNF) family of cytokines can induce apoptosis by binding to their specific receptors on target cells. Nagata, supra; Baker, S. J. and Reddy, E. P., Oncogene 12:1-9, 1996. The receptors for the TNF family of cytokines belong to a family of proteins referred to as the TNFR family, which is characterized by an extracellular domain of highly conserved cysteine residues contained in cysteine-rich pseudorepeats (Chaudhary et al., Immunity 7:821-830, 1997). In addition, several members of the TNFR family possess a conserved cytoplasmic domain of approximately 80 amino acids called the death domain, which functions to initiate an intracellular apoptotic signaling cascade upon binding of the appropriate cytokine. (See Chaudhary et al., supra:; Walczak et al., EMBO J. 16:5386-5397, 1997.) TNFR proteins containing death domains comprise a death receptor subfamily which includes: TNFR1 (Tartiglia et al., Cell 74:845-853, 1993); Fas (also called CD95 and Apo-1) (Itoh and Nagata, J. Biol. Chem. 268:10932-10937, 1993); death receptor 3 (DR3, also called TRAMP, Apo-3, Wsl-1, and LARD) (Chinnaiyan et al., Science 274:990-992,1996; Kiston et al., Nature 384:372-375, 1996); TRAIL-R1 (also known as DR4) (Pan et al., Science 276:111-113, 1997); and TRAIL-R2 (also called DR5) (Pan et al., Science 277:815-818, 1997). The death domains of these proteins are shown in FIG. 1.
Fas, the most studied death receptor, is expressed on the surface of most cell types, including epithelial cells, fibroblasts, T and B cells, liver hepatocytes and some tumor cells (Nagata, Nature Medicine 2:1306-1307, 1996; French et al., Nature Medicine 3:387-388, 1997). However, FasL is primarily expressed by activated leukocytes of the immune system, including cytotoxic T lymphocytes (CTL's) and natural killer (NK) cells (Nagata, Cell, supra). It is believed that the Fas ligand (FasL) plays a role in the immune response of these cells to induce apoptosis in target cells expressing Fas. Such target cells include virus-infected cells and tumor cells. On the other hand, leukocytes also express Fas, which can result in down regulation of the immune response due to activated leukocytes killing each other (Nagata, Cell, supra).
Recently, it was discovered that FasL is also expressed in immune-privileged sites such as the eye chamber, parts of the nervous system, and testis and it is believed that any activated leukocytes entering such sites are immediately killed through the FasL-Fas apoptotic pathway, thereby preventing a potentially crippling immune response (Nagata, Cell, supra). This finding could potentially be applied to preventing transplant rejection and, indeed, one group has reported that islet allografts were protected from immune rejection by cotransplantation with syngeneic myoblasts expressing functional FasL (Lau et al., Science 273:109-112, 1996).
The discovery of FasL expression in immune-privileged sites led a number of groups to examine whether the means by which tumor cells avoid destruction is through expression of FasL. A number of tumor cell types were subsequently reported to constitutively express FasL, including Iymphoma and leukemia cells (Tanake, et al., Nature Med. 2:317-322, 1996) various nonlymphoid carcinoma cells, including colon cancer (O'Connell, et al., J. Exp. Med. 184:1075-1082, 1996), hepatocellular carcinoma (Strand et al., Nature Med. 21361-1366, 1996) and melanoma (Hahne et al., Science 274:1363-1366, 1996). As a result of expressing FasL, many tumor cells have the ability to kill attacking CTL and NK cells thereby reducing the immune response against the tumor. In addition, it has been reported that some types of tumors become resistant to Fas-mediated apoptosis, either by downregulation of Fas expression or by other unknown mechanisms, and thereby avoid being killed by the infiltrating leukocytes (Nagata, Nat. Med., supra.; Strand et al., supra; Hahne et al., supra). Because alterations in Fas-FasL regulation, including upregulation of FasL expression and downregulation of Fas expression, may be involved in tumor cells avoiding destruction by the immune system, it would be desirable to devise an approach that would reduce the effect of such changes in Fas-FasL regulation. In one such approach it was recently reported that the anti-cancer drug doxorubicin enhances expression of both Fas and Fasl in tumor cells (Friesen et al., Nature Med. 2:574-577, 1996).
Recent reports have associated other disease states with dysfunction of the Fas system, including hypereosinophilic syndromes in humans (Lenardo et al., J. Exp. Med. 183:721-724, 1996), hepatitis (Kondo et al., Nat. Med. 3:409-413, 1997) and the autoimmune disease Hashimoto's thyToiditis (HT) (Giordano et al., Science 175:960-963, 1997). Consequently, it has been suggested that inappropriate upregulation of Fas may be a causal factor in other autoimmune diseases involving tissues which constitutively express FasL (French et al., supra).
Human adenoviruses (used interchangeably herein with Ad), which cause disease in the respiratory tract, conjunctiva, intestine, urinary tract and liver, have evolved elaborate mechanisms to overcome host antiviral defenses, including at least four of the seven known proteins encoded by the early region 3 (E3) transcription unit which have been reported to inhibit the host immune response to Ad-infected cells (Fejer et al., J. Virol. 68:5871-5881, 1994; Sparer et al., J. Virol. 770:2431-2439, 1996). One of these proteins is a 19 kDa glycoprotein (gp19K), which inhibits CTL-mediated lysis of Ad-infected cells in vitro (Efrat et al., Proc. Natl. Acad. Sci. 92:6947-6951, 1995). Three other E3 proteins, the 14.7K protein and 10.4K protein in combination with the 14.5K protein (referenced hereinafter as the 10.4K/14.5K complex), protect adenovirus-infected cells against cytolysis and the inflammatory response induced by tumor necrosis factor-α (TNF-α) both in vitro and in vivo (Sparer et al., supra; Krajcsi et al., J. Virol. 70:4904-4913, 1996; Dimitrov et al., J. Virol. 71:2830-2837, 1997). Although the exact stoichiometry of 10.4K and 14.5K proteins in this complex is not known, it is believed to consist of one 14.5K polypeptide in physical association with a dimer formed by full-length and short forms of the 10.4K polypeptide joined in disulfide linkage. Stewart et al, supra.
Efrat et al. have reported that the expression of the one of the Ad E3 genes, i.e. the gene encoding the 19 kDa glycoprotein (gp19K), can prolong survival of pancreatic islet allografts. The islets were obtained from transgenic animals prepared to contain the entire E3 genomic DNA from human Ad, however, the gp19K mRNA was prominently expressed with little or no expression of the 10.4K protein which makes up a portion of the 10.4/14.5 complex. The islet allografts survived reportedly due to the expression of the gp19K protein and there was no suggestion in this reference that the 10.4K or 14.5K proteins either separately or in the 10.4K/14.5K complex played any role in the survival of the allografts.
Nevertheless, the 10.4/14.5 complex can protect Ad-infected cells from the inflammatory response in the context of Ad infection (Sparer et al., supra) and, although it has not been heretofore recognized, it is possible that the 10.4K/14.5K complex could also provide a novel basis for modulating the immune system in certain disease processes.